There exist a strong interest in identifying specific cell types in an effort to gain enriched populations of the cells. Having possession of an enriched population may allow for a better understanding of the specific cell types or even provide uses in various situations including transplantation, gene therapy, treatment of disease including cancers such as leukaemias, neoplastic cancers including breast cancers, or repair of tissues and skin.
Stem cells give rise to cells, which ultimately contribute to various parts of the plant or animal. One type of stem cell is the haematopoietic stem cell.
Hematopoietic cells are responsible for an extraordinarily diverse range of activities. They are divided into several lineages, including lymphoid, myeloid and erythroid. The lymphoid lineage, comprising B cells and T cells, produces antibodies, regulates cellular immunity, and detects foreign agents such as disease-causing organisms in the blood. The myeloid lineage, which includes monocytes, granulocytes, and megakaryocytes, monitors the blood for foreign bodies, protects against neoplastic cells, scavenges foreign materials, and produces platelets. The erythroid lineage includes red blood cells, which carry oxygen.
Haematopoietic stern cells are capable of self-renewal, multilineage proliferation and differentiation, and long-term support of the haematopoietic and lymphoid systems. They form a subpopulation within the haematopoietic progenitor compartment (HPC), which mainly comprises cells of more limited potentiality. HPC cells are mainly located within the bone marrow stroma, where complex interaction with stromal cells, extracellular matrix components and cytokines, permits regulation of cell proliferation and differentiation. HPC cells are also present in the blood under a variety of physiological, pathological and iatrogenic circumstances. HPC can be harvested from bone marrow or peripheral blood, and will re-engraft the bone marrow following intravenous infusion in patients who have received ablative (i.e. destructive) doses of chemotherapy and/or radiotherapy, leading to regeneration of haematopoiesis and immunity. Thus, HPC cell transplantation is of considerable clinical utility in the management of patients with haematological and solid malignancies, bone marrow failure, and inborn errors of haematopoiesis, immunity or metabolism.
There is thus a need for supplies of autologous HPC cells which may be cultured in vitro prior to reintroduction into a patient whose HPC cell population has been depleted due to chemotherapy and/or radiotherapy. The populations of such HPC cells may take many weeks or months to recover naturally to their normal levels. The use of autologous cells from the patient themselves avoids rejection of the transplanted cells.
In vivo, HPC cells are generally located within the bone marrow stroma. In vitro, HPC cells are able to adhere to bone marrow stromal layers before proliferating and releasing more committed progenitors. Stem cells undergo marked proliferation and differentiation into multiple lineages, ultimately giving rise to fully differentiated cells or progeny, such as red blood cells, platelets, a variety of white blood cells, and also immune cells such as T lymphocytes and B lymphocytes. Thus, the reintroduction of HPC cells or stem cells into the patient who is depleted therein, allows efficient repopulation of these haematopoietic cell types.
The relative paucity of hematopoietic stem cells has prevented extensive research on stem cells and hematopoietic differentiation in general. The ready availability of a cell population enriched in hematopoietic stem cells would make possible the identification of biological modifiers affecting stem cell behavior. For example, there may be as yet undiscovered growth factors associated with (1) early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the ability to control stem cell proliferation.
The availability of sufficient numbers of stem cells in an enriched population would also be extremely useful, for example, in reconstituting hematopoiesis in patients undergoing treatments which destroy stem cells, such as cancer chemotherapy.
Considerable evidence supports the proposal that the localization of hemopoiesis to the bone marrow (BM) in adult mammals involves developmentally regulated interactions between primitive hematopoietic stem cells (HSC) and the stromal cell mediated hemopoietic microenvironment (HM) of the marrow.
Anatomical location of maturing hemopoietic cells within the BM is better understood than the spatial distribution of more primitive cells. Previous studies in the mouse have established that lineage restricted clonogenic hemopoietic progenitor cells (HPC) also conform to a well-defined spatial distribution across the axis of the femur with greatest numbers near the central longitudinal vein. In contrast, hierarchically more primitive progenitors, colony-forming unit spleen (CFU-S), exhibit the converse distribution with low numbers in the central region of the marrow and greatest enrichment in a region adjacent to bone the endosteum.
The reestablishment of hemopoiesis by intravenously infused cells requires several coordinated events including homing, migration and lodgement of HPC within the BM HM. The initial event, homing, is the specific recruitment of circulating HSC to the BM and involves the selective recognition of HSC by the microvascular endothelium of the marrow and trans-endothelial cell migration into the extravascular hemopoietic space. In contrast, lodgement encompasses events following extravasation and is defined as the selective migration of cells to suitable HM niches in the extravascular compartment. Current data suggests that homing involves a similar cascade of cell adhesion molecules (CAMs) to those participating in the extravasation of mature leukocytes into tissues. Primitive hemopoietic cells exhibit a broad repertoire of CAMs including various members of the integrin, sialomucin, Ig super family and CD44 families. Current data suggest key roles for the sialomucin receptor for P-selectin, PSGL-1, the β1 integrin VLA-4 and the receptor for SDF-1, CXCR4 in HSC homing to the BM. In contrast, very little is known about the molecules that influence the site of HSC lodgement following homing to the BM.
However, identification of these specific cell types by cell surface markers has generally proven to be the best means of identification. The identification of additional cell surface antigens would clearly be of major value in the identification, isolation and further characterization of hematopoietic stem cells.
Until recently, it has not been possible to define the spatial distribution of hemopoietic stem cells (HSC) within the BM. This is due to the rarity of HSC and the lack of a single, unique antigenic marker allowing their unambiguous identification in situ.
Accordingly, it is an object of the present invention to overcome or at least alleviate some of the problems of the prior art.